HK40074953A - Humanized nanobodies against novel coronaviruses and use thereof - Google Patents

Description

Anti-novel coronavirus humanized nano antibody and application thereof

Technical Field

The invention belongs to the technical field of antibodies. More particularly, relates to an anti-novel coronavirus humanized nano antibody and application thereof.

Background

The successful application of monoclonal antibodies in cancer detection and biological targeted therapy has led to a revolution in tumor therapy. However, the traditional monoclonal antibody has too high molecular weight (150kD) and is difficult to penetrate tissues, so that the effective concentration of a tumor region is low, and the treatment effect is insufficient; in addition, the traditional antibody has high immunogenicity, and the modified antibody is difficult to reach the original affinity, so that the wide application of the traditional antibody in clinic is limited.

Nanobodies are the smallest antibody molecules at present, with molecular weights 1/10 of common antibodies. The nano antibody not only has the antigen reactivity of the monoclonal antibody, but also has some unique functional characteristics, such as small molecular weight, strong stability, good solubility, easy expression, weak immunogenicity, strong penetrability, strong targeting property, simple humanization, low preparation cost and the like, and almost perfectly overcomes the defects of long development period, low stability, harsh storage conditions and the like of the traditional antibody.

The new coronavirus (SARS-COV-2) has been widely spread around the world since the outbreak in 2019, causing severe economic loss, social burden and other negative effects; in addition, various novel coronavirus variants are discovered in succession, wherein the variants refer to the fact that mutation or deletion of certain base of a certain gene occurs on the basis of the genome of the original virus, the mutation or the deletion of the base can cause the property of the virus to change, and the virus can change infectivity, host range, transmission strength, virulence, pathogenicity, the severity of disease, prognosis and immunogenicity. This leads to changes in the clinical overall pathogenesis and epidemiology, and even to possible effects on immunogenicity and prevention of immunity.

The broad-spectrum micromolecule antiviral drugs such as Rudexiwei and the like developed at present have certain curative effect on the novel coronavirus wild strain, but have no curative effect and specificity on the novel coronavirus wild strain, the Delta variant strain and the Omicron variant strain, and are lack of novel coronavirus special-effect drugs clinically; therefore, there is a need to develop a drug having specific therapeutic effects on both wild strains and mutant strains of novel coronavirus.

Disclosure of Invention

The invention aims to provide a novel anti-coronavirus single domain antibody, wherein the amino acid sequence of the single domain antibody is shown as any one of SEQ ID NO. 5-14.

It is another object of the present invention to provide a humanized nanobody against a novel coronavirus, which comprises the single domain antibody. The humanized nano antibody has low immunogenicity risk, can effectively block the combination of novel coronavirus SARS-COV-2RBD protein of wild type, Delta variant and Omicron variant and human ACE2 receptor protein, and has obvious novel coronavirus neutralizing activity.

It is another object of the present invention to provide a nucleic acid encoding the single domain antibody or the humanized nanobody.

The invention also provides an expression vector carrying the nucleic acid.

The invention also provides a host cell carrying said nucleic acid, comprising said expression vector, or capable of expressing said single domain antibody, or capable of expressing said humanized nanobody.

The invention also provides a pharmaceutical composition containing the single domain antibody, the humanized nanobody, the nucleic acid, the expression vector or the host cell.

The invention also provides application of the single-domain antibody, the humanized nano-antibody, the nucleic acid, the expression vector and the host cell in preparation of a medicine for treating and/or preventing novel coronaviruses.

The present invention also provides a method for treating and/or preventing a novel coronavirus, which comprises administering an effective dose of the pharmaceutical composition to a subject infected with the novel coronavirus.

Drawings

FIG. 1 is a diagram of a plasmid containing a desired gene.

FIG. 2 is the result of the neutralizing activity of the humanized nanobody on the novel coronavirus SARS-COV-2RBD protein of the wild strain.

FIG. 3 is the result of the neutralizing activity of humanized nanobody on novel coronavirus SARS-COV-2RBD protein of Delta variant.

FIG. 4 is a graph showing the results of the neutralization activity of humanized nanobody on novel coronavirus SARS-COV-2RBD protein of Omicron variant.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The invention provides a novel coronavirus resistant single domain antibody, wherein the amino acid sequence of the single domain antibody is shown as any one of SEQ ID NO 5-14.

In some embodiments, the amino acid sequence of the single domain antibody is set forth in SEQ ID NO 10.

The invention provides an anti-novel coronavirus humanized nanobody, which comprises the single-domain antibody.

In the present invention, the term "nanobody" refers to a heavy chain antibody (e.g., derived from a camel body) lacking a light chain, and a single domain antibody obtained by cloning the variable region thereof is a minimum functional antigen-binding fragment and has a relative molecular mass (Mr) of only about 15000. The nano antibody has the characteristics of small molecular weight, strong stability, good solubility, easy expression, low immunogenicity and the like.

The invention also includes variants, derivatives and analogs of the nanobodies. As used herein, the terms "variant," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as a nanobody of the invention. A polypeptide variant, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a pro-polypeptide sequence, or a fusion polypeptide). Such variants, derivatives and analogs are within the scope of those skilled in the art as defined herein.

In addition, other amino acid sequences which do not substantially influence the activity, the expression quantity and the stability of the nano antibody can be added at the amino terminal or the carboxyl terminal of the nano antibody. These added amino acid sequences may facilitate expression (e.g., signal peptide), purification (e.g., 6 × His sequence), or other sequences that may facilitate the activity, expression or stability of the nanobody.

In some embodiments, the humanized nanobody further comprises a half-life extending domain.

In a preferred embodiment, the half-life extending domain is selected from an immunoglobulin Fc region or a serum albumin binding domain.

In a preferred embodiment, the half-life extending domain is an immunoglobulin Fc region.

In a preferred embodiment, the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO 15.

In some embodiments, the humanized nanobody has an amino acid sequence as set forth in any one of SEQ ID NOS 16-25.

In a preferred embodiment, the amino acid sequence of the humanized nanobody is shown in SEQ ID NO 21.

The invention also provides a nucleic acid encoding the single domain antibody or the humanized nanobody.

Nucleic acids are typically RNA or DNA, and nucleic acid molecules can be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acid is used when it is ligated to a vector.

The invention also provides an expression vector carrying the nucleic acid.

In the present invention, the term "vector" includes plasmids, expression vectors, cloning vectors, viral vectors and the like. Various carriers known in the art may be used. For example, an expression vector can be formed by selecting a commercially available vector and then operably linking a nucleotide sequence encoding the nanobody of the present invention to an expression control sequence.

The invention also provides a host cell carrying said nucleic acid, comprising said expression vector, or capable of expressing said single domain antibody, or capable of expressing said humanized nanobody.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include E.coli, Bacillus subtilis, and the like. The host cells for expressing the nanobody include escherichia coli, yeast cells, insect cells, COS cells, CHO cells, and the like. After obtaining the transformed host cell, the cell may be cultured under conditions suitable for expression of the nanobody of the present invention, thereby expressing the nanobody; then separating out the expressed nano antibody.

The invention also provides a pharmaceutical composition containing the single domain antibody, the humanized nanobody, the nucleic acid, the expression vector or the host cell.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In the present invention, the "pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Specifically, the solvent may be any one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, and the like, and a combination thereof. Of course, a "pharmaceutically acceptable carrier" may also include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which serve to prolong the shelf life or effectiveness of the antibody.

In addition, the application of the single domain antibody, the humanized nano antibody, the nucleic acid, the expression vector and the host cell in the preparation of the medicine for treating and/or preventing the novel coronavirus is also within the protection scope of the invention.

The present invention also provides a method for treating and/or preventing a novel coronavirus, which comprises administering an effective dose of the pharmaceutical composition to a subject infected with the novel coronavirus.

The invention has the following beneficial effects:

the invention provides a humanized nano antibody for resisting a novel coronavirus, which has low immunogenicity risk, can effectively block the combination of novel coronavirus SARS-COV-2RBD protein of wild type, Delta variant and Omicron variant and human ACE2 receptor protein, and has obvious novel coronavirus neutralizing activity; therefore, the humanized nano antibody, the nucleic acid, the expression vector and the host cell provided by the invention have good application prospects in preparation of medicines for treating and/or preventing novel coronaviruses.

Embodiments of the present invention will be described in detail with reference to examples.

Example 1 humanization design of anti-novel coronavirus humanized Nanobodies

Carrying out humanized design on the novel anti-camel-derived nano antibody (CN 202210089563.7, wherein the amino acid sequences of CDR1, CDR2 and CDR3 are shown as SEQ ID NO: 1-3 in sequence, the amino acid sequence of VHH is shown as SEQ ID NO:4, and the number of VHH is RX011), and carrying out humanized modification by a CDR-grafting method; firstly, carrying out homologous modeling on the camel-derived nano antibody by using MOE software, and combining the empirical analysis of an inventor to form key composition amino acid residues which can influence the stable conformation of an antigen binding region according to a model structure; then, searching a human immunoglobulin database, searching a human IGVH sequence with high homology with the camel-derived nano antibody in a human germline antibody library as a humanized transformation template, respectively comparing the camel-derived nano antibody with a matched human IGVH sequence, analyzing key composition amino acid residues which can influence the conformational stability of an antigen binding region on the camel-derived nano antibody and sites which are inconsistent with the human IGVH sequence in an emphatic analysis mode by combining the experience of an inventor, and observing whether humanized replacement can be carried out or not in the model; according to different humanized substitution degrees, the VHH amino acid sequences of 10 humanized nano antibodies against the novel coronavirus are designed, the VHH numbers are RX012-RX021 in sequence, and the specific amino acid sequences are shown in Table 1:

SEQ ID NO:1：RCTFNWDG

SEQ ID NO:2：ISWSGQEP

SEQ ID NO:3：AAAQYTGASYSILRDQVGYDY

SEQ ID NO:4：

QVQLVESGGGPVQAGGSLRLSCTCSRCTFNWDGMGWFRQAPGKEREFVATISWSGQEPAYADSVKGRFTISRDKPKNTVYLQMTSLKSEDTAVYYCAAAQYTGASYSILRDQVGYDYWGQGTRVTVSA

TABLE 1

For ease of validation, the huFc constant region was chosen as the expression tag, with the specific amino acid sequence shown in table 2:

TABLE 2

Example 2 preparation of humanized Nanobodies against novel coronavirus

Protein transient expression:

as shown in FIG. 1, a plasmid containing a target gene is introduced into a host cell Expi293 after forming a cationic complex with PEI, and the foreign gene on the plasmid is transcribed and translated in the cell while the plasmid is in the cell, thereby obtaining the target protein.

Expi293 cells were grown at 37 deg.C, 8% carbon dioxide, 130rpm and 2E6 cells were seeded into 1L shake flasks at approximately 300mL prior to transfection by cell counting. Preparation of transfection complexes preparation for transfection: firstly, 750 mu g of target plasmid is added into a 50mL centrifuge tube containing 15mL Opti-MEM reagent, and the mixture is gently mixed and marked as tube A; adding 1.5mg of PEI as a transfection reagent into a 50mL centrifuge tube containing 15mL of Opti-MEM reagent, gently mixing uniformly, and then incubating at room temperature for 5min, wherein the tube is marked as a tube B; and dropwise adding the PEI diluent of the tube B into the DNA diluent of the tube A, slightly and uniformly mixing, incubating at room temperature for 15min, adding the PEI-target plasmid compound into the Expi293 cells after the incubation is finished, and placing the cells in a shaking table at 37 ℃ for continuous culture. The samples were collected until after D7-D10.

And (3) purifying a compound sample:

the transient cell expression solution was centrifuged at 9000rpm/20min, and the supernatant was collected and sterile-filtered through a 0.22 μm filter. The purification was performed by ProA affinity chromatography. The process is as follows, using an AKTA avant 150 chromatographic apparatus, with at least 5CV of equilibration buffer (10mM PBS) to equilibrate a chromatography column (e.g., MabSelectSuRe LX, GE), loading the sample onto the column, allowing the target protein to adsorb onto the column while other impurities are separated by breakthrough. After the loading was completed, the column was washed again with at least 5CV of equilibration buffer (10mM PBS), followed by elution of the target protein with elution buffer (20mM NaAc, pH 3.4), and a neutralization buffer (1M Tris, pH8.0) was added to the collection tube in advance, the volume of the neutralization buffer added being determined according to the estimated content of the eluted sample, and typically 10% of the elution volume was added.

The antibody was prepared by conventional methods and the expression supernatant was purified by ProA affinity chromatography. The process is as follows, using an AKTA avant 150 chromatographic apparatus, with at least 5CV of equilibration buffer (10mM PBS) to equilibrate a chromatography column (e.g., MabSelectSuRe LX, GE), loading the sample onto the column, allowing the target protein to adsorb onto the column while other impurities are separated by breakthrough. After the loading was completed, the column was washed again with at least 5CV of equilibration buffer (10mM PBS), followed by elution of the target protein with elution buffer (20mM NaAc, pH 3.4), and a neutralization buffer (1M Tris, pH8.0) was added to the collection tube in advance, the volume of the neutralization buffer added being determined according to the estimated content of the eluted sample, and typically 10% of the elution volume was added.

The sample is subjected to concentration measurement by using Biotek-Epoch-Take-3, the antibody concentration is detected by using an A280 method, namely, the extinction coefficient E.C. is 1.37 (predicted according to an amino acid sequence), the optical path is 0.05mm (the optical paths of different holes of a Take-3 plate are slightly different and can be automatically corrected), the light absorption value of the sample is detected by using equipment, and the concentration of the antibody to be detected is calculated according to Lambert-Beer law. If the sample concentration is too low, ultrafiltration concentration is required, and an ultrafiltration concentration tube (Ultra-15 Centrifugal Filter Devices, 30kD) following the general protocol provided in the specification, the sample concentration was concentrated to > 0.5 mg/mL; and collecting a concentrated end sample, sterilizing and filtering the concentrated end sample by using a 0.22um sterile needle filter (Cobalter, PES, 0.22um and the diameter of 13mm), and subpackaging and freezing for later use.

The VHH amino acid sequence RX012-RX021 of the 10 anti-new coronavirus humanized nano-antibodies is directly connected with hIgG1-Fc (SEQ ID NO:15) to obtain the anti-new coronavirus humanized nano-antibody, and the serial numbers of the anti-new coronavirus humanized nano-antibodies are sequentially R1383-R1392; RX011 is directly connected with hIgG1-Fc (SEQ ID NO:15) to obtain camel-derived nano-antibody with the number of R1382.

TABLE 3

The expression level and purity of the humanized nanobody against the novel coronavirus are shown in table 4, and the results show that the expression level and purity data of the humanized antibody are ideal.

TABLE 4

Example 3 neutralizing Activity of humanized Nanobodies against wild-type Strain of novel coronavirus

The neutralizing activity of the humanized nano antibody to the wild strain novel coronavirus SARS-COV-2RBD protein is detected by using a competition method, R1382 in the example 2 is used as a control group, and the specific experimental steps are as follows:

coating conditions are as follows:

viral protein: 2ug/mL of wild strain novel coronavirus SARS-COV-2RBD protein;

coating liquid: 50mm pH9.51 CB;

coating volume: 100 ul/well;

coating temperature: 2-8 ℃;

coating time: 18 hours;

sealing liquid: 1% BSA +1 XPBS;

sealing temperature: 37 ℃;

sealing time: 3 hours;

sample adding: 50uL of the antibody to be tested (all antibodies initially at 5ug/mL and serially diluted in 5-fold gradient) was added, incubated for 30min, washed 5 times with plate wash (1 XPBS), added with 50uL of ACE2 protein, washed 3 times with plate wash (1 XPBS), and subjected to chromogenic detection.

The results of the activity of neutralizing the wild-type novel coronavirus SARS-COV-2RBD protein by the humanized nanobody are shown in Table 5 and FIG. 2, and the results show that the humanized nanobody prepared in example 2 can effectively block the combination of the wild-type SARS-COV-2RBD protein and the human ACE2 receptor protein, and has the activity of neutralizing the novel coronavirus; among them, R1383, R1384, R1385, R1386, R1387 and R1388 show significant neutralizing activity against wild-type novel coronaviruses.

TABLE 5

Example 4 neutralizing Activity of humanized Nanobodies against novel coronavirus of Delta variant

The neutralizing activity of humanized nano antibody R1383-R1392 to novel coronavirus SARS-COV-2RBD protein of Delta variant is detected by using a competition method, and the concrete experimental steps are the same as those in example 3 by using R1382 in example 2 as a control group.

The results of the activity of the humanized nanobody in neutralizing the novel SARS-COV-2RBD protein of the Delta variant strain are shown in Table 6 and FIG. 3, and the results show that the humanized nanobody prepared in example 2 can effectively block the combination of the novel SARS-COV-2RBD protein of the Delta variant strain and the human ACE2 receptor protein, and has the novel coronavirus neutralizing activity; among them, R1385, R1386, R1387, R1388, R1389 showed significant novel coronavirus neutralizing activity of Delta variants.

TABLE 6

Numbering	Delta variant novel coronavirus SARS-COV-2 IC50
		R1382	0.01369nM
R1383	0.02232nM
		R1384	0.02133nM
R1385	0.01237nM
		R1386	0.009197nM
R1387	0.01038nM
		R1388	0.007211nM
R1389	0.01255nM
		R1390	0.01807nM
R1391	0.0204nM
		R1392	0.02351nM

Example 5 neutralizing Activity of humanized Nanobody against novel coronavirus of Omicron variant

The neutralizing activity of humanized nanobody R1383-R1392 against novel coronavirus SARS-COV-2RBD protein of Omicron variant strain was examined by competition method, and R1382 in example 2 was used as a control group, and the detailed experimental procedure was the same as that in example 3.

The results of the activity of the humanized nanobody in neutralizing the novel coronavirus SARS-COV-2RBD protein of the Omicron variant are shown in Table 7 and FIG. 4, and the results show that the humanized nanobody prepared in example 2 can effectively block the combination of the novel coronavirus SARS-COV-2RBD protein of the Omicron variant and the human ACE2 receptor protein, and has the novel coronavirus neutralizing activity; among them, R1385, R1386, R1387, R1388 and R1389 showed significant activity of neutralizing novel coronavirus of an Omicron variant.

TABLE 7

Numbering	Omicron variant coronavirus SARS-COV-2 IC50
		R1382	0.17nM
R1383	0.7067nM
		R1384	0.8007nM
R1385	0.1458nM
		R1386	0.08825nM
R1387	0.1157nM
		R1388	0.1174nM
R1389	0.122nM
		R1390	0.2232nM
R1391	0.2169nM
		R1392	0.2369nM

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Guangdong Fengcong pharmaceutical Co Ltd

<120> anti-novel coronavirus humanized nano antibody and application thereof

<130> 2022

<160> 25

<170> PatentIn version 3.5

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<213> Artificial sequence

<400> 14

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln

100 105 110

Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 15

<211> 232

<212> PRT

<213> Artificial sequence

<400> 15

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 16

<211> 360

<212> PRT

<213> Artificial sequence

<400> 16

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Cys Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Ala Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln

100 105 110

Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 17

<211> 360

<212> PRT

<213> Artificial sequence

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Cys Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Ala Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln

100 105 110

Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 18

<211> 360

<212> PRT

<213> Artificial sequence

<400> 18

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Cys Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln

100 105 110

Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 19

<211> 360

<212> PRT

<213> Artificial sequence

<400> 19

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln

100 105 110

Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 20

<211> 360

<212> PRT

<213> Artificial sequence

<400> 20

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln

100 105 110

Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 21

<211> 360

<212> PRT

<213> Artificial sequence

<400> 21

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln

100 105 110

Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 22

<211> 360

<212> PRT

<213> Artificial sequence

<400> 22

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln

100 105 110

Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 23

<211> 360

<212> PRT

<213> Artificial sequence

<400> 23

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln

100 105 110

Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 24

<211> 360

<212> PRT

<213> Artificial sequence

<400> 24

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Pro Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln

100 105 110

Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

<210> 25

<211> 360

<212> PRT

<213> Artificial sequence

<400> 25

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Cys Ser Arg Cys Thr Phe Asn Trp Asp

20 25 30

Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Thr Ile Ser Trp Ser Gly Gln Glu Pro Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Thr Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Ala Gln Tyr Thr Gly Ala Ser Tyr Ser Ile Leu Arg Asp Gln

100 105 110

Val Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

130 135 140

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

145 150 155 160

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

165 170 175

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

180 185 190

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

195 200 205

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

210 215 220

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

225 230 235 240

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

245 250 255

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

260 265 270

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

275 280 285

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

290 295 300

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

305 310 315 320

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

325 330 335

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

340 345 350

Ser Leu Ser Leu Ser Pro Gly Lys

355 360

Claims (10)

1. The novel coronavirus-resistant single-domain antibody is characterized in that the amino acid sequence of the single-domain antibody is shown as any one of SEQ ID NO 5-14.

2. Humanized nanobody against a novel coronavirus, characterized in that it comprises a single domain antibody according to claim 1.

3. The humanized nanobody of claim 2, further comprising a half-life extending domain;

preferably, the half-life extending domain is selected from an immunoglobulin Fc region or a serum albumin binding domain.

4. The humanized nanobody of claim 3, wherein the amino acid sequence of the Fc region of immunoglobulin is shown in SEQ ID NO 15.

5. The humanized nanobody according to any one of claims 2 to 4, wherein the amino acid sequence of the humanized nanobody is represented by any one of SEQ ID NO 16 to 25.

6. A nucleic acid encoding the single domain antibody of claim 1 or the humanized nanobody of any one of claims 2 to 5.

7. An expression vector carrying the nucleic acid of claim 6.

8. A host cell carrying the nucleic acid of claim 6, comprising the expression vector of claim 7, or capable of expressing the single domain antibody of claim 1, or capable of expressing the humanized nanobody of any one of claims 2 to 5.

9. A pharmaceutical composition comprising the single domain antibody of claim 1, the humanized nanobody of any one of claims 2 to 5, the nucleic acid of claim 6, the expression vector of claim 7 or the host cell of claim 8.

10. Use of the single domain antibody of claim 1, the humanized nanobody of any one of claims 2 to 5, the nucleic acid of claim 6, the expression vector of claim 7, the host cell of claim 8 for the preparation of a medicament for the treatment and/or prevention of a novel coronavirus.